The overall goal of these studies is to elucidate the membrane trafficking pathway that is used in the recycling of synaptic vesicles (SVs) in living synapses. According to previous data, SV recycling could either involve two sequential budding events, a primary budding from the plasma membrane and a secondary budding from endosomes, or only a single budding step from the plasma membrane. It has not been possible to distinguish between these two possibilities in previous studies, primarily due to difficulties to determine whether internal "endosome-like" structures seen in ultrathin sections represent endosomes or plasma membrane invaginations. These difficulties can be overcome by using high voltage electron microscopic (HVEM) examination of thicker sections combined with electron tomography to determine precisely 3D relationships between structures. We will apply HVEM to examine the 3D ultrastructure of presynaptic membrane structures in specimen blocks (1-2 5m semithin sections) that include the entire synaptic region of the giant reticulospinal synapse in lamprey. This synapse has been extensively characterized and proved suitable as a model to study vesicle recycling mechanisms. By applying high frequency action potential stimulation, the synaptic vesicle pool can be depleted. The recovery of the vesicles will be examined in specimens fixed at different time points following cessation of stimulation. To visualize putative endosomal intermediates that may be difficult to observe under physiological conditions, we will use protocols that perturb synaptic vesicle endocytosis at different steps. These include microinjection of proteins and nucleotides that interfere with the fission of coated pits, and perfusion of calcium-free solution which arrests the onset of the formation of clathrin coated pits. The application of HVEM and tomographic reconstruction to the lamprey model will make it possible to detail the microanatomy of synaptic vesicle recycling and thereby to elucidate a membrane trafficking pathway that is of key importance in synaptic transmission. Work on this project is underway.